METHOD FOR EFFICIENTLY PRODUCING ß MYOSIN HEAVY CHAIN IN CARDIAC MUSCLE CELLS DIFFERENTIATED FROM INDUCED PLURIPOTENT STEM CELLS DERIVED FROM HOMO SAPIENS

ABSTRACT

The present invention provides a method for producing a β myosin heavy chain in cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens. In the present method, first, a liquid culture medium containing the cardiac muscle cells is supplied onto a substrate comprising a first electrode, a second electrode and insulative fibers on the surface thereof. At least a part of the insulative fibers is located between the first electrode and the second electrode in a top view of the substrate. Then, the substrate is left at rest. Finally, the cardiac muscle cells are cultivated, while a pulse electric current is applied to the cardiac muscle cells through the first electrode and the second electrode.

CROSS-REFERENCE OF RELATED APPLICATIONS

This application is a Divisional application of the patent applicationSer. No. 15/848,020, filed on Dec. 20, 2017, which claims the benefit ofJapanese Application No. 2017-039998, filed on Mar. 3, 2017, the entiredisclosures of which applications are incorporated by reference herein.

INCORPORATION BY REFERENCE-SEQUENCE LISTING

The material contained in the ASCII text file named“P1006798US01_ST25.txt” created on Nov. 22, 2017, and having a file sizeof 18, 746 bytes is incorporated by reference herein.

BACKGROUND 1. Technical Field

The present invention relates to a method for efficiently producing a βmyosin heavy chain in cardiac muscle cells differentiated from inducedpluripotent stem cells derived from Homo sapiens.

2. DESCRIPTION OF THE RELATED ART

Japanese patent application laid-open publication No. Sho 60-110287discloses that cell proliferation is promoted by application of electricpulse to the cultivated cells.

Japanese patent application laid-open publication No. Hei 4-141087discloses a method that cells are differentiated by application ofelectric voltage to the cells through a liquid culture medium.

U.S. Pat. No. 8,916,189 discloses a cell culture support for formingstring-shaped cardiomyocyte aggregates.

Japanese patent application laid-open publication No. 2013-188173discloses a method for creating cell tissue having function.

United States Patent Application Publication No. 2015/0017718 disclosesa method for inducing cardiac differentiation of a pluripotent stemcell.

WO 2016/060260 discloses a method for producing a tissue fragment,particularly a myocardial tissue fragment which contains cultured cellshaving an oriented configuration. See FIG. 4B, FIG. 9A, and paragraphs0055, 0131, 0141, 0142, and 0153 thereof.

SUMMARY

The present invention provides a method for producing a β myosin heavychain in cardiac muscle cells differentiated from induced pluripotentstem cells derived from Homo sapiens, the method comprising:

(a) supplying a liquid culture medium containing the cardiac musclecells onto a substrate comprising a first electrode, a second electrodeand insulative fibers on the surface thereof to coat a surface of thefirst electrode, a surface of the second electrode, and an regionbetween the first electrode and the second electrode with the cardiacmuscle cells;

wherein

at least a part of the insulative fibers is located between the firstelectrode and the second electrode in a top view of the substrate; and

an angle formed between each of not less than 90% of the insulativefibers and an imaginary straight line which passes through both thefirst electrode and the second electrode is not more than ±20 degrees inthe top view;

(b) leaving the substrate at rest; and

(c) cultivating the cardiac muscle cells, while a pulse electric currentis applied to the cardiac muscle cells through the first electrode andthe second electrode.

The present invention provides a method for efficiently producing a βmyosin heavy chain in cardiac muscle cells differentiated from inducedpluripotent stem cells derived from Homo sapiens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a top view of a substrate.

FIG. 2 shows an enlarged view of a region A included in FIG. 1.

FIG. 3 shows a graph showing an example of desirable pulse electriccurrent.

FIG. 4 shows a top view of the substrate in one step included in amethod for fabricating the substrate.

FIG. 5 shows an enlarged view of a region B included in FIG. 4.

FIG. 6A shows an enlarged top view of an end part of an electric wiring.

FIG. 6B shows a cross-sectional view taken along the line 6B-6B includedin FIG. 6A.

FIG. 7A shows an enlarged top view of the end part of the electricwiring.

FIG. 7B shows a cross-sectional view taken along the line 7B-7B includedin FIG. 7A.

FIG. 8A shows a cross-sectional view of the substrate on which a liquidculture medium has been supplied.

FIG. 8B shows a cross-sectional view of the substrate on which a liquidculture medium has been supplied.

FIG. 9A is a fluorescent microscope photograph of the cardiac musclecells in the inventive example 1.

FIG. 9B is a fluorescent microscope photograph of the cardiac musclecells in the comparative example 2.

FIG. 9C is a fluorescent microscope photograph of the cardiac musclecells in the comparative example 4.

FIG. 9D is a fluorescent microscope photograph of the cardiac musclecells in the comparative example 6.

FIG. 10A shows an enlarged top view of the end part of the electricwiring in the comparative examples 2 and 3.

FIG. 10B shows a cross-sectional view taken along the line 10B-10Bincluded in FIG. 10A.

FIG. 11A shows an enlarged top view of the end part of the electricwiring in the comparative examples 4 and 5.

FIG. 11B shows a cross-sectional view taken along the line 11B-11Bincluded in FIG. 11A.

FIG. 12A shows an enlarged top view of the end part of the electricwiring in the comparative examples 6 and 7.

FIG. 12B shows a cross-sectional view taken along the line 12B-12Bincluded in FIG. 12A.

FIG. 13A is a microscope photograph of a first electrode, a secondelectrode, and an insulative fibers which have been formed on thethus-provided substrate in the inventive example 1.

FIG. 13B is another microscope photograph of the first electrode, thesecond electrode, and the insulative fibers which have been formed onthe substrate in the inventive example 1.

FIG. 13C is a microscope photograph of the first electrode, the secondelectrode, and the insulative fibers which have been formed on thesubstrate 100 used in the comparative example 2 and the comparativeexample 3.

FIG. 13D is a microscope photograph of the first electrode, the secondelectrode, and the insulative fibers which have been formed on theprovided substrate used in the comparative example 4 and the comparativeexample 5.

DETAILED DESCRIPTION OF THE EMBODIMENT

Hereinafter, an embodiment of the present invention will be describedwith reference to the drawings.

As disclosed in FIG. 2C of United States Patent Application PublicationNo. 2015/0017718, an amount of production of a β myosin heavy chain(hereinafter, referred to as “β MHC”) is significantly smaller incardiac muscle cells differentiated from induced pluripotent stem cellsderived from Homo sapiens than in cardiac muscle cells included in aliving body. The β MHC is one kind of polypeptides providing support fora structure of the cell. For the maturation of the cardiac muscle cellsdifferentiated from induced pluripotent stem cells derived from Homosapiens, it is important to produce the β MHC efficiently.

The β MHC has a primary structure consisting of the amino acid sequencerepresented by the following SEQ ID NO: 1.

(SEQ ID NO: 1) MGDSEMAVFGAAAPYLRKSEKERLEAQTRPFDLKKDVFVPDDKQEFVKAKIVSREGGKVTAETEYGKTVTVKEDQVMQQNPPKFDKIEDMAMLTFLHEPAVLYNLKDRYGSWMIYTYSGLFCVTVNPYKWLPVYTPEVVAAYRGKKRSEAPPHIFSISDNAYQYMLTDRENQSILITGESGAGKTVNTKRVIQYFAVIAAIGDRSKKDQSPGKGTLEDQIIQANPALEAFGNAKTVRNDNSSRFGKFIRIHFGATGKLASADIETYLLEKSRVIFQLKAERDYHIFYQILSNKKPELLDMLLITNNPYDYAFISQGETTVASIDDAEELMATDNAFDVLGETSEEKNSMYKLTGAIMHFGNMKFKLKQREEQAEPDGTEEADKSAYLMGLNSADLLKGLCHPRVKVGNEYVTKGQNVQQVIYATGALAKAVYERMENWMVTRINATLETKQPRQYFIGVLDIAGFEIFDFNSFEQLCINFTNEKLQQFFNHHMFVLEQEEYKKEGIEWTFIDFGMDLQACIDLIEKPMGIMSILEEECMFPKATDMTFKAKLFDNHLGKSANFQKPRNIKGKPEAHFSLIHYAGIVDYNIIGWLQKNKDPLNETVVGLYQKSSLKLLSTLFANYAGADAPIEKGKGKAKKGSSFQTVSALHRENLNKLMTNLRSTHPHFVRCIIPNETKSPGVMDNPLVMHQLRCNGVLEGIRICRKGFPNRILYGDFRQRYRILNPAAIPEGQFIDSRKGAEKLLSSLDIDHNQYKFGHTKVFFKAGLLGLLEEMRDERLSRIITRIQAQSRGVLARMEYKKLLERRDSLLVIQWNIRAFMGVKNWPWMKLYFKIKPLLKSAEREKEMASMKEEFTRLKEALEKSEARRKELEEKMVSLLQEKNDLQLQVQAEQDNLADAEERCDQLIKNKIQLEAKVKEMNERLEDEEEMNAELTAKKRKLEDECSELKRDIDDLELTLAKVEKEKHATENKVKNLTEEMAGLDEIIAKLTKEKKALQEAHQQALDDLQAEEDKVNTLTKAKVKLEQQVDDLEGSLEQEKKVRMDLERAKRKLEGDLKLTQESIMDLENDKQQLDERLKKKDFELNALNARIEDEQALGSQLQKKLKELQARIEELEEELESERTARAKVEKLRSDLSRELEEISERLEEAGGATSVQIEMNKKREAEFQKMRRDLEEATLQHEATAAALRKKHADSVAELGEQIDNLQRVKQKLEKEKSEFKLELDDVTSNMEQIIKAKANLEKMCRTLEDQMNEHRSKAEETQRSVNDLTSQRAKLQTENGELSRQLDEKEALISQLTRGKLTYTQQLEDLKRQLEEEVKAKNALAHALQSARHDCDLLREQYEEETEAKAELQRVLSKANSEVAQWRTKYETDAIQRTEELEEAKKKLAQRLQEAEEAVEAVNAKCSSLEKTKHRLQNEIEDLMVDVERSNAAAAALDKKQRNFDKILAEWKQKYEESQSELESSQKEARSLSTELFKLKNAYEESLEHLETFKRENKNLQEEISDLTEQLGSSGKTIHELEKVRKQLEAEKMELQSALEEAEASLEHEEGKILRAQLEFNQIKAEIERKLAEKDEEMEQAKRNHLRVVDSLQTSLDAETRSRNEALRVKKKMEGDLNEMEIQLSHANRMAAEAQKQVKSLQSLLKDTQIQLDDAVRANDDLKENIAIVERRNNLLQAELEELRAVVEQTERSRKLAEQELIETSERVQLLHSQNTSLINQKKKMDADLSQLQTEVEEAVQECRNAEEKAKKAITDAAMMAEELKKEQDTSAHLERMKKNMEQTIKDLQHRLDEAEQIALKGGKKQLQKLEARVRELENELEAEQKRNAESVKGMRKSERRIKELTYQTEEDRKNLLRLQDLVDKLQLKVKAYKRQAEEAEEQANTNLSKFRKVQHELDEAEERADIAESQVNKLRAKSRDIGTKGLNEE

For reference, myosin regulatory light chain 2 (hereinafter, referred toas “MYL2”) is also produced in the cardiac muscle cells. The MYL2 has aprimary structure consisting of the amino acid sequences represented bythe following SEQ ID NO: 2.

(SEQ ID NO: 2) MAPKKAKKRAGGANSNVFSMFEQTQIQEFKEAFTIMDQNRDGFIDKNDLRDTFAALGRVNVKNEEIDEMIKEAPGPINFTVFLTMFGEKLKGADPEETILNAFKVFDPEGKGVLKADYVREMLTTQAERFSKEEVDQMFAAFPPDVTGNL DYKNLVHIITHGEEKD

Hereinafter, the cardiac muscle cells differentiated from inducedpluripotent stem cells derived from Homo sapiens are just referred to as“cardiac muscle cells”. As well known, the induced pluripotent stemcells may be referred to as “iPS cells”.

(Step (a))

First, a liquid culture medium containing cardiac muscle cells aresupplied on a substrate 100 comprising a first electrode, a secondelectrode, and insulative fibers on the surface thereof.

FIG. 1 shows a top view of the substrate 100. FIG. 2 shows an enlargedview of a region A included in FIG. 1.

As shown in FIG. 1, the substrate 100 comprises a glass base 1 and anenclosure 10 located on the glass base 1. The surface of the glass base1 is provided with electric contacts 2 and electric wirings 3. Each ofthe electric contacts 2 is connected to one end of one electric wiring3. Within the enclosure 10, an insulative sheet 60 is disposed on theglass base 1. The electric wirings 3 are covered with the insulativesheet 60.

As shown in FIG. 2, other ends of the electric wirings 3 are exposed.The exposed parts function as a first electrode 31 and a secondelectrode 32. In FIG. 2, four electric wirings 3 are drawn. The firstelectrode 31 is formed of the exposed end part of the electric wiring 3located on the left. Similarly, the second electrode 32 is formed of theexposed end part of the electric wiring 3 located on the right.

As shown in FIG. 1 and FIG. 2, insulative fibers 50 are disposed on thesurface of substrate 100. The fibers 50 are required to be insulative.This is because a short circuit is prevented from being formederroneously between the first electrode 31 and the second electrode 32.In case where the short circuit is formed erroneously, a pulse electriccurrent which will be described later fails to be applied to the cardiacmuscle cells.

As shown in FIG. 2, at least a part of the insulative fibers 50 islocated between the first electrode 31 and the second electrode 32. Incase where the insulative fibers 50 are not located between the firstelectrode 31 and the second electrode 32 (including a case where noinsulative fibers 50 are provided on the substrate 100), the β MHC isnot produced efficiently, as demonstrated in the comparative example 6which will be described later.

The insulative fibers 50 are exposed on the surface of the substrate100. The first electrode 31 and the second electrode 32 are also exposedon the surface of substrate 100.

The insulative fibers 50 have orientation such that an angle formedbetween each of not less than 90% of the insulative fibers 50 and animaginary straight line which passes through both the first electrode 31and the second electrode 32 is not more than ±20 degrees in the top viewof substrate 100. In other words, each of the not less than 90% of theinsulative fibers 50 forms an angle of not more than 20 degrees withregard to the imaginary straight line. Therefore, not less than 90% ofthe insulative fibers 50 are substantially parallel to a direction of anelectric field generated when an electric current (e.g., pulse electriccurrent) is caused to flow between the first electrode 31 and the secondelectrode 32. Needless to say, the imaginary straight line does notexist actually on the substrate 100. Desirably, the angle is not morethan ±5 degrees. See the paragraph 0023 of U.S. patent application Ser.No. 15/519,341, which is incorporated herein by reference.

In case where less than 90% of the insulative fibers 50 aresubstantially parallel to the imaginary straight line which passesthrough both the first electrode 31 and the second electrode 32, the βMHC is not produced efficiently. See the comparative examples 3-6 whichwill be described later. In the comparative examples 2-3, almost all ofthe insulative fibers 50 are substantially perpendicular to theimaginary straight line which passes through both the first electrode 31and the second electrode 32. In other words, in the comparative examples2-3, each of the almost all of the insulative fibers 50 forms an angleof approximately 90 degrees with regard to the imaginary straight line.In the comparative examples 4-5, a roughly half of the insulative fibers50 are perpendicular to the imaginary straight line which passes throughboth the first electrode 31 and the second electrode 32, and the otherroughly half of the insulative fibers 50 are parallel to the imaginarystraight line.

Desirably, each of the insulative fibers 50 has a diameter of not lessthan 1 micrometer and not more than 5 micrometers. It is desirable thatthe material of the insulative fibers 50 is selected from the groupconsisting of polystyrene, polycarbonate, polymethylmethacrylate,polyvinyl chloride, polyethylene terephthalate, polyamide,polymethylglutarimide, or polylactic acid. It is desirable that thedistance between the first electrode 31 and the second electrode 32 isnot less than 150 micrometers and not more than 5,000 micrometers.

One example of a fabrication method of the substrate 100 will bedescribed in more detail in the examples which will be described later.A skilled person who has read the examples which will be described laterwould understand easily the fabrication method of the substrate 100.

As shown in FIG. 8A, a liquid culture medium 182 containing cardiacmuscle cells 180 is supplied to the surface of the above-mentionedsubstrate 100. The liquid culture medium 182 is spread onto the surfaceof the substrate 100 within the enclosure 10. In this way, the surfaceof the first electrode 31, the surface of the second electrode 32, and aregion C between the first electrode 31 and the second electrode 32 arecoated with the cardiac muscle cells. In case where at least one of thesurface of the first electrode 31, the surface of the second electrode32, and the region C fails to be coated with the cardiac muscle cells,the pulse electric current fails to be applied to the cardiac musclecells 180 in the step (b) which will be described later. As a result,the β MHC fails to be produced efficiently. As just described, in thestep (a), the liquid culture medium 182 containing the cardiac musclecells 180 having an amount sufficient to coat the surface of the firstelectrode 31, the surface of the second electrode 32, and the region Cis supplied to the surface of substrate 100.

(Step (b))

The Step (b) is conducted out after the step (a). In the Step (b), thesubstrate 100 is left at rest. In this way, the cardiac muscle cellsadhere on the insulative fibers 50 or the surface of substrate 100.Desirably, the substrate 100 is left at rest over 24 hours.

(Step (c))

The Step (c) is conducted after the step (b). In the step (c), while apulse electric current is applied to the cardiac muscle cells 180through the first electrode 31 and the second electrode 32, the cardiacmuscle cells 180 are cultivated. The same pulse electric current may beapplied to the first electrode 31 and the second electrode 32. When thepulse electric current is applied to the first electrode 31 and thesecond electrode 32, a reference electrode 4 may be used. The referenceelectrode 4 is grounded. As shown in FIG. 8A, the reference electrode 4may be provided on the surface of the substrate 100. However, as shownin FIG. 8B, the reference electrode 4 is not necessary to be provided onthe surface of the substrate 100. In FIG. 8B, the reference electrode 4is included in the inside of the liquid culture medium 182. Anyway, itis desirable that the reference electrode 4 is in contact with theliquid culture medium 182.

FIG. 3 is a graph showing an example of a desirable pulse electriccurrent. As shown in FIG. 3, the desirable pulse electric current has aperiod of 333 milliseconds to 2 seconds (1 second in FIG. 3). One pulseis either positive or negative. In FIG. 3, first, a negative pulse isapplied, and then a positive pulse is applied. While the negative pulseis applied, an electric current flows from the cardiac muscle cells tothe first electrode 31 (or the second electrode 32). While the positivepulse is applied, an electric current flows from the first electrode 31(or the second electrode 32) to the cardiac muscle cells.

One pulse has a time length of 0.05 milliseconds to 4 milliseconds (0.4milliseconds in FIG. 3) and a height (namely, an electric current value)of 1 microampere-20 microamperes (3-12 microamperes, in FIG. 3). It isdesirable that the size of the pulse (namely, an area of one pulse inFIG. 3) is not less than 0.1 nano coulomb and not more than 1.0 nanocoulomb. More desirably, the rate of the size of the pulse to the areaof the first electrode 31 (or the second electrode 32) is not less than0.04 coulombs/square meter and not more than 0.4 coulombs/square meter.It is desirable that the size of the negative pulse (namely, the area ofthe negative pulse in FIG. 3) is the same as the size of the positivepulse (namely, the area of the positive pulse in FIG. 3).

As demonstrated in the inventive example 1, the thus-cultivated cardiacmuscle cells 180 contain a lot of β MHC. In other words, the β MHC isproduced efficiently in the thus-cultivated cardiac muscle cells 180. Incase where the pulse electric current fails to be applied, the β MHCfails to be produced efficiently. Seethe comparative examples 1, 3, 5,and 7 which will be described later.

EXAMPLES

Hereinafter, the present invention will be described in more detail withreference to the following examples.

(Fabrication of Substrate 100)

The substrate 100 shown in FIG. 1 was fabricated as below. First, theglass base 1 having a shape of a square was prepared. The glass base 1had a thickness of 0.7 millimeters and an area of approximately 2500square millimeters (i.e., 50 millimeters×50 millimeters). Then, as shownin FIG. 4, the electric contacts 2 and the electric wirings 3 wereformed on the glass base 1. The electric wirings 3 were formed byetching an indium tin oxide film having a thickness of 150 nanometersusing a photoresist. The number of the electric contacts 2 and theelectric wirings 3 was sixty-eight.

Then, the surface of the glass base 1 was coated with an insulation film40 consisting of a photosensitive acrylic acid resin. The electriccontacts 2 were not coated with the insulation film 40. Each one end ofthe electric wirings 3 was not coated with the insulation film 40, sincethe one end of the electric wiring 3 was used as the first electrode 31,the second electrode 32, or the reference electrode 4. Subsequently, theglass base 1 was subjected to plasma surface treatment at an RF power of18 W for two minutes with a plasma treatment apparatus (available fromHarrick Plasma Company, trade name: “PDC-32G”).

FIG. 5 shows an enlarged view of a region B included in FIG. 4. Oneelectrode set 6 consisted of the ends of the four electric wirings 3, asshown in FIG. 5. The number of the electrode set 6 was 16 sets. The endsof remaining four electric wirings 3 were used for the referenceelectrode 4. FIG. 6A shows an enlarged top view of the end part of theelectric wiring 3. FIG. 6B shows a cross-sectional view taken along theline 6B-6B included in FIG. 6A.

The end of the electric wiring 3 exposed on the surface (i.e., the firstelectrode 31 and the second electrode 32) had a size of approximately 15micrometers×approximately 170 micrometers. The reference electrode 4 hadan area of approximately 200 square micrometers. The distance betweenthe ends of adjacent two electric wirings 3 was approximately 400micrometers. The distance of adjacent two electrode sets 6 wasapproximately 4 millimeters.

Meanwhile, insulative fibers made of polymethyl glutaric imide wereformed on the surface of an aluminum tape (available from HitachiMaxell. Ltd., trade name: SLIONTEC) by an electrospinning method inaccordance with the process disclosed in the paragraph 0122 of U.S.patent application Ser. No. 15/519,341. Unlike the process disclosed inthe paragraph 0122 of U.S. patent application Ser. No. 15/519,341, anejection time of polymethyl glutaric imide in the electrospinning methodwas 30 minutes in the inventive example 1. The insulative fibers had asurface coverage of 30%.

Then, the aluminum tape having the insulative fibers was disposed on thesurface of the glass base 1 so that the insulative fibers weresandwiched between the aluminum tape and the electric wiring 3. Thealuminum tape having the insulative fibers was impressed onto thesurface of the insulation film 40 and the exposed ends of the electricwirings 3. Then, the aluminum tape was removed. FIG. 7A shows anenlarged top view of the end part of the electric wiring 3. FIG. 7Bshows a cross-sectional view taken along the line 7B—7B included in FIG.7A. As shown in FIG. 7A and FIG. 7B, the insulative fibers 50 weretranscribed on the surface of the insulation film 40 and the exposedends of the electric wirings 3. As shown in FIG. 2 and FIG. 7A, not lessthan 90% of the insulative fibers 50 were disposed in a directionparallel to the imaginary straight line which passes through the firstelectrode 31 and the second electrode 32 (namely, in a horizontaldirection in the figures).

Then, as shown in FIG. 2, a silicone resin sheet 60 (available fromToray Dow Corning company, trade name: SYLGARD 184) was adhered on theinsulation film 40 with a silicone adhesive. The silicone resin sheet 60had a thickness of approximately 1 millimeter. The ends of the electricwirings 3 and their peripheries were not coated with the silicone resinsheet 60. Furthermore, the enclosure 10 was adhered with the siliconeadhesive so as to include the silicone resin sheet 60 in the insidethereof. The enclosure 10 was formed of glass. The enclosure 10 had aninternal diameter of approximately 22 millimeters, an external diameterof approximately 25 millimeters, and a height of approximately 10millimeters.

The exposed ends of the electric wirings 3 were plated with platinumblack 5. Specifically, the parts were plated at a current density of 20mA/cm² for two minutes using a plating solution. During the plating, theelectric wirings 3 were used as cathodes. The plating solution had thecomposition shown in Table 1. The first electrode 31 or the secondelectrode 32 was formed through such plating on the surface of the endof the electric wiring 3. In other words, the first electrode 31 and thesecond electrode 32 were formed of platinum black.

TABLE 1 Composition Chemical formula Concentration HexachloroplatinicH₂PtCl₆ · 6H₂O    1% (IV) acid Lead acetate (CH₃COO)2Pb · 3H₂O  0.01%Hydrochloric acid HCl 0.0025%

In this way, the substrate 100 was provided. FIG. 13A is a microscopephotograph of the first electrode 31, the second electrode 32, and theinsulative fibers 50 which have been formed on the thus-providedsubstrate 100. FIG. 13B is also a microscope photograph of the firstelectrode 31, the second electrode 32, and the insulative fibers 50which have been formed on the substrate 100 provided similarly. As shownin FIG. 13B, a small amount of non-oriented fibers are included in theinsulative fibers 50 due to the problem in the fabrication process bythe electrospinning method. The amount of the non-oriented fibers isless than 10%.

(Cultivation of Cardiac Muscle Cells)

Using the substrate 100, cardiac muscle cells differentiated by inducedpluripotent stem cells derived from Homo sapiens were cultivated. Andthen, production ratio of the β MHC was measured. Specifically, cardiacmuscle cells differentiated by induced pluripotent stem cells derivedfrom Homo sapiens (available from iPS Academia Japan, Inc., trade name:iCell Cardiomycytes) were used. Pursuant to the protocol described inthe manual attached to iCell Cardiomycytes, a liquid culture mediumcontaining cardiac muscle cells differentiated by induced pluripotentstem cells derived from Homo sapiens was prepared.

Then, as shown in FIG. 8A, the liquid culture medium 182 was suppliedonto the substrate 100. The density of the cardiac muscle cells 180 onthe substrate 100 was 1.5×10⁴/square millimeter. In this way, thesurface of the first electrode 31, the surface of the second electrode32, and the region C were coated with the cardiac muscle cells 180. Thecardiac muscle cells 180 was cultivated pursuant to the protocoldescribed in the manual attached to iCell Cardiomycytes.

Two days after the supply of the liquid culture medium 182, the pulseelectric current shown in FIG. 3 is applied with the reference electrode4 to the cardiac muscle cells 180 through the first electrode 31 and thesecond electrode 32 shown in FIG. 2 to stimulate the cardiac musclecells 180. For the application of the pulse electric current, a pulseelectric current generator 200 was electrically connected to the firstelectrode 31 and the second electrode 32 through the electric contacts2. The electric potential of the liquid culture medium 182 wasmaintained at standard electric potential (i.e., GND) through thereference electrode 4.

The pulse electric current was applied to the cardiac muscle cells 180for 12 days, except in time of a change of a culture medium. In thisway, the cardiac muscle cells 180 were cultivated.

(Measurement of Production Ratio of β MHC)

The production ratio of the β MHC contained in the thus-cultivatedcardiac muscle cells 180 was measured as below.

The cardiac muscle cells were fixed with 4% paraformaldehyde and werepermeabilized in phosphate buffered saline (PBS) plus 0.5% Triton X-100for 0.5 hours. After blocking in a 5% normal donkey serum, 3% BSA, and0.1% Tween 20 in PBS for 16 hours at 4 degrees Celsius, the cells wereincubated for 16 hours at 4 degrees Celsius with mouse MYH7 monoclonalIgM primary antibodies (available from Santa Cruz Biotechnology, tradename: SC-53089) diluted at 1:100 with a blocking buffer. In this way,the primary antibodies were bound to the cardiac muscle cells. Theantigen capable of binding to the primary antibody was β MHC (GenBank:AAA51837.1).

Then, the cardiac muscle cells to which the primary antibodies werebound were washed with PBS. Subsequently, the cardiac muscle cells wereincubated for 1 hour at 25 degrees Celsius with fluorescently-labelledanti-mouse IgM secondary antibodies (available from JacksonImmunoresearch labs., trade name: DyLight-594-Donkey anti-mouse IgM)diluted at 1:1,000 with the blocking buffer. In this way, thefluorescently-labelled secondary antibodies were bound to the primaryantibodies. In this way, the cardiac muscle cells were fluorescentlylabelled.

The fluorescently-labelled cardiac muscle cells were observed using afluorescent microscope. FIG. 9A is a fluorescent microscope photographof the cardiac muscle cells in the inventive example 1. The brightnessof the observed fluorescence was converted into 256 gradation digitalbrightness level. Digital brightness level 0 means that brightness islowest. Digital brightness level 255 means that brightness is highest.

Hereinafter, the β MHC production ratio is defined as a rate of the sumof the areas of the regions each having a digital brightness level ofnot less than 65 to the area of the whole of the observation region. Inother words, the βMHC production ratio is calculated according to thefollowing mathematical formula.

(β MHC Production Ratio)=(Sum of Areas of the regions each having adigital brightness level of not less than 65)/(Area of the whole of theobservation region)

In the inventive example 1, the β MHC production ratio was 57.9%.

For reference, production ratio of myosin regulatory light chain 2(hereinafter, referred to as “MYL2”) contained in the cultivated cardiacmuscle cells was measured similarly. In particular, the MYL2 productionratio was calculated similarly to the case of the β MHC productionratio, except for the following two matters.

(I) In place of the mouse MYH7 monoclonal IgM antibodies, rabbit MYL2polyclonal IgG antibodies (dilution ratio: 1/200, available fromProteintech Company, trade name: 109060-1-AP) was used as the primaryantibodies.

(II) In place of the anti-mouse IgM fluorescently-labelled secondaryantibodies, anti-rabbit IgG fluorescently-labelled antibodies (availablefrom Jackson Immunoresearch labs., trade name: Alexa Fluor 488 Donkeyanti-rabbit IgG) was used as the secondary antibodies.

As a result, the MYL2 production ratio was 36.7% in the inventiveexample 1.

Comparative Example 1

An experiment similar to the inventive example 1 was conducted, exceptthat no pulse electric current was applied.

Comparative Example 2

An experiment similar to the inventive example 1 was conducted, exceptthat almost all of the insulative fibers 50 were disposed substantiallyperpendicularly (namely, in a vertical direction in FIG. 10A) to theimaginary straight line which passes through the first electrode 31 andthe second electrode 32, as shown in FIG. 10A and FIG. 10B. FIG. 9B is afluorescent microscope photograph of the cardiac muscle cells in thecomparative example 2. FIG. 13C is a microscope photograph of the firstelectrode 31, the second electrode 32, and the insulative fibers 50which have been formed on the thus-obtained substrate 100 used in thecomparative example 2 and the comparative example 3 which will bedescribed later. As shown in FIG. 13C, in the comparative examples 2-3,the insulative fibers 50 were disposed in a direction perpendicular tothe imaginary straight line which passes through the first electrode 31and the second electrode 32 (namely, in the vertical direction in thefigure).

Comparative Example 3

An experiment similar to the inventive example 1 was conducted, exceptthat almost all of the insulative fibers 50 were disposed substantiallyperpendicularly (namely, in a vertical direction in FIG. 10A) to theimaginary straight line which passes through the first electrode 31 andthe second electrode 32, as shown in FIG. 10A and FIG. 10B, and exceptthat no pulse electric current was applied.

Comparative Example 4

An experiment similar to the inventive example 1 was conducted, exceptthat roughly half of the insulative fibers 50 were disposed parallel(namely, in the horizontal direction in FIG. 11A) to the imaginarystraight line which passes through the first electrode 31 and the secondelectrode 32 and the other roughly half of the insulative fibers 50 weredisposed perpendicularly (namely, in a vertical direction in FIG. 11A)to the imaginary straight line, as shown in FIG. 11A and FIG. 11B. FIG.9C is a fluorescent microscope photograph of the cardiac muscle cells inthe comparative example 4. FIG. 13D is a microscope photograph of thefirst electrode 31, the second electrode 32, and the insulative fibers50 which have been formed on the thus-obtained substrate 100 used in thecomparative example 4 and the comparative example 5 which will bedescribed later. As shown in FIG. 13D, in the comparative examples 4-5,roughly half of the insulative fibers 50 (ejection time: 15 minutes)were disposed in a direction parallel to the imaginary straight linewhich passes through the first electrode 31 and the second electrode 32(namely, in the horizontal direction in the figure), whereas the otherroughly half of the insulative fibers 50 (ejection time: 15 minutes)were disposed in a direction perpendicular to the imaginary straightline (namely, in the vertical direction in the figure).

Comparative Example 5

An experiment similar to the inventive example 1 was conducted, exceptthat some of the insulative fibers 50 were disposed parallel (namely, inthe horizontal direction in FIG. 11A) to the imaginary straight linewhich passes through the first electrode 31 and the second electrode 32and the other insulative fibers 50 were disposed perpendicularly(namely, in a vertical direction in FIG. 11A) to the imaginary straightline, as shown in FIG. 11A and FIG. 11B, and except that no pulseelectric current was applied.

Comparative Example 6

An experiment similar to the inventive example 1 was conducted, exceptthat no insulative fibers 50 were disposed, as shown in FIG. 12A andFIG. 12B. FIG. 9D is a fluorescent microscope photograph of the cardiacmuscle cells in the comparative example 6.

Comparative Example 7

An experiment similar to the inventive example 1 was conducted, exceptthat no insulative fibers 50 were disposed, as shown in FIG. 12A andFIG. 12B, and except that no pulse electric current was applied.

The following Table 2 shows the β MHC production rate measured in theinventive example 1 and the comparative examples 1-7.

TABLE 2 Relation Between Direction of Insulative Pulse β MHC fibers andDirection of electric production Electric Field current rate (%)  I. E.1 FIG. 13A or FIG. 13B Applied 57.9  C. E. 1 FIG. 13A or FIG. 13B No14.5  C. E. 2 FIG. 13C Applied 31.9  C. E. 3 FIG. 13C No 10.3  C. E. 4FIG. 13D Applied 36.5  C. E. 5 FIG. 13D No 15.8  C. E. 6 No insulativefibers Applied 15.4  C. E. 7 No insulative fibers No 9.8 “I. E.” means“Inventive Example”. “C. E.” means “Comparative Example”. “ElectricField” means the electric field generated between the first electrode 31and the second electrode 32 by the electric current pulse.

The following Table 3 shows the MYL2 production rate measured in theinventive example 1 and the comparative examples 1-7.

TABLE 3 Relation Between Direction of Insulative Pulse MYL2 fibers andDirection of electric production Electric Field current rate (%)  I. E.1 FIG. 13A or FIG. 13B Applied 36.7 C. E. 1 FIG. 13A or FIG. 13B No 25.1C. E. 2 FIG. 13C Applied 30.0 C. E. 3 FIG. 13C No 19.0 C. E. 4 FIG. 13DApplied 32.5 C. E. 5 FIG. 13D No 24.0 C. E. 6 No insulative fibersApplied 16.2 C. E. 7 No insulative fibers No 10.1

As is clear from Table 2, when both of the following requirements (I)and (II) are satisfied, the β MHC production rate is a significantlyhigh value of 57.9%. See the inventive example 1.

Requirement (I): The insulative fibers 50 have orientation such that anangle formed between each of not less than 90% of the insulative fibers50 and an imaginary straight line which passes through both the firstelectrode 31 and the second electrode 32 is not more than ±20 degrees inthe top view.

Requirement (II): The cardiac muscle cells 180 are cultivated, while thepulse electric current is applied thereto.

On the other hand, in case where at least one of the requirements (I)and (II) fails to be satisfied, the β MHC production rate is a low valueof less than 36.5%. See the comparative examples 1-7.

As is clear from Table 3, regardless to the direction of the insulativefibers, the MYL2 production rate is a constant value of approximately32%-37%. On the other hand, as is clear from Table 1, the β MHCproduction rate is significantly increased, when both of therequirements (I) and (II) are satisfied. In other words, the use of theinsulative fibers increases the production amount of polypeptide(including protein) in the cardiac muscle cells. Among the polypeptideproduced in the cardiac muscle cells, when both of the requirements (I)and (II) are satisfied, the β MHC is produced at the significantly highproduction rate, unlike other polypeptide such as MYL2.

INDUSTRIAL APPLICABILITY

The present invention provides a method for efficiently producing 0myosin heavy chain in cardiac muscle cells differentiated from inducedpluripotent stem cells derived from Homo sapiens.

REFERENTIAL SIGNS LIST

-   100 Substrate-   1 Glass plate-   2 Electric contact-   3 Electric wiring-   4 Reference electrode-   5 Platinum black-   6 Electrode set-   10 Enclosure-   31 First electrode-   32 Second electrode-   40 Insulation film-   50 Insulative fiber-   60 Insulative sheet-   A Region-   B Region-   C Region-   180 Cardiac muscle cells-   182 Liquid culture medium-   200 Pulse electric current generator

1. A substrate comprising: a first electrode; a second electrode; andinsulative fibers, wherein the first electrode, the second electrode,and the insulative fibers are provided on a surface of the substrate; atleast a part of the insulative fibers is located between the firstelectrode and the second electrode in a top view of the substrate; andan angle formed between each of not less than 90% of the insulativefibers and an imaginary straight line which passes through both thefirst electrode and the second electrode is not more than ±20 degrees inthe top view.
 2. The substrate according to claim 1, further comprisinga reference electrode on the surface thereof.